The ability to generate induced pluripotent stem cells (iPSCs) without a permanent DNA sequence change is extremely important for many subsequent applications. A useful strategy is to integrate the genes necessary for transformation into the target genome using the DNA cut & paste piggyBac transposon (1) as the vector. The piggyBac transposon has a large cargo size and a high integration efficiency. The piggyBac transposase promotes insertion of the piggyBac transposon into TTAA target sites by binding to specific sequences at the transposon ends. Upon integration, the element becomes stably associated with the host genome and can serve as a long-term source of transcription factors required for cell transformation. Once iPS cell transformation has occurred, the piggyBac vector can then be re-exposed to transposase and excised by its natural “precise excision” pathway, in which the insertion site is restored to its pre-transposon TTAA sequence (2, 3). To avoid further genome modification, it is important that the excised transposon not re-integrate.
We are interested in developing improved piggyBac transposons as more useful tools for generating transgene-free iPSCs. A particularly useful tool for removal of the piggyBac vector after iPS cells transformation would be a piggyBac transposase that can promote excision at high frequency but is defective for re-integration following excision, i.e., an Exc+Int− transposase.
Using in vitro and in vivo assays in mammalian cells (4), we have isolated a piggyBac Exc+Int− transposase. In Saccharomyces cerevisiae (4), and confirmed in mammalian cells, we have also isolated a transposase that is hyperactive for excision but still integration negative, Exc+hyperInt−.